Journal: Cell reports
Article Title: FANCM promotes PARP inhibitor resistance by minimizing ssDNA gap formation and counteracting resection inhibition.
doi: 10.1016/j.celrep.2024.114464
Figure Lengend Snippet: Figure 1. Depletion of FANCM, but not FANCB, induces hypersensitivity to PARPis (A) Schematic diagram of the construction of an HCT116 cell line with FANCM C-terminally tagged with a mAID and a GFP. Positions of primers p1, p2, and p3 designed to confirm bi-allelic tagging are shown. (B) Cells were treated with Dox and IAA for 48 h. Depletion of FANCM was verified by western blotting. (C–E) Clonogenic cell survival assays were carried out on HCT116FANCM-mAID-GFP cells treated with or without Dox and IAA and the indicated concentrations of cisplatin, olaparib, and veliparib. (F) Cells depleted of FANCM or FANCB by siRNAs were fractionated to separate soluble and chromatin fractions. The fractions were analyzed by western blotting for the indicated proteins. (G–I) Clonogenic cell survival assays were carried out on mock-depleted, FANCM-depleted, and FANCB-depleted cells. They were treated with indicated concentrations of cisplatin, olaparib, and veliparib. Data in (C)–(E) and (G)–(I) are represented as mean ± SD, n = 3 independent experiments. p values were determined using an unpaired two-tailed t test. See also Figure S1.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Zeocin Thermo Fisher Cat# R25001 Puromycin Thermo Fisher Cat# A11138-02 Geneticin Thermo Fisher Cat# 10131035 SuperSignal West Pico PLUS Chemiluminescent Substrate Thermo Fisher Cat# 34580 Colcemid Thermo Fisher Cat# 15212012 Insulin, human recombinant Thermo Fisher Cat# 12585014 Hydrocortisone Sigma-Aldrich Cat# H4001 5-Chloro-20-deoxyuridine thymidine analog, CldU Sigma-Aldrich Cat# C6891 5-Iodo-2 -deoxyuridine, IdU Sigma-Aldrich Cat# 17125 Nocodazole Sigma-Aldrich Cat# M1404 5-Ethynyl-20-deoxyuridine, EdU Cayman Cat# 20518 Etoposide Sigma-Aldrich Cat# 341205 Olaparib Cayman Cat# 10621 Veliparib Cayman Cat# 11505 Methyl methanesulfonate Sigma-Aldrich Cat# 129925 Hydroxyurea Sigma-Aldrich Cat# H8627 S1 Nuclease Thermo Fisher Cat# EN0321 Protease and Phosphatase Inhibitor Thermo Fisher Cat# A32961 Protease Inhibitor Tablets Thermo Fisher Cat# A32965 Q5 High-Fidelity DNA polymerase New England Biolabs Cat#M0491S Critical commercial assays NucleoSpin Plasmid Transfection-grade, Mini kit Macherey-Nagel Cat# 740490.250 NucleoBond Xtra Midi kit, transfection-grade Macherey-Nagel Cat# 740410.50 GeneJET Gel Extraction Kit Thermo Fisher Cat# K0691 DNA Ligation Kit, Mighty Mix TaKaRa Cat# 6023 CloneJET PCR Cloning Kit Thermo Fisher Cat# K1231 Subcellular Protein Fractionation Kit for Cultured Cells Thermo Fisher Cat# 78840 Experimental models: Cell lines HCT116 ATCC Cat# CCL-247 HCT116: OsTIR1 Previous study99 N/A HCT116: OsTIR1 FANCM-mAID-GFP This paper N/A HCT116: OsTIR1 FANCM-mAID-GFP 53BP1 KO This paper N/A HCT116: OsTIR1 FANCM-mAID-GFP PARP1 KO This paper N/A 293 DR-GFP gift from Stephen West N/A MCF-7 ATCC Cat# HTB-22 SUM149PT gift from Stephen West N/A Oligonucleotides See Table S1 N/A N/A Recombinant DNA Plasmid: pMK289 (mAID-mClover-NeoR) Addgene Cat# 72827 Plasmid: pMK289 (mAID-mClover-BleoR) Previous study99 N/A Plasmid: pMK243 (Tet-OsTIR1-PURO) Addgene Cat# 72835 Plasmid: pX330-U6-Chimeric_BB-cBh-hSpCas9 Addgene Cat# 42230 Plasmid: pX330-sgPRIMPOL This paper N/A Plasmid: pMA-RQ FANCM This paper N/A Plasmid: pMA-RQ FANCM-mAID-Clover (Zeo) This paper N/A Plasmid: pMA-RQ FANCM-mAID-Clover (Neo) This paper N/A Plasmid: pX330 sgFANCM C terminus This paper N/A Plasmid: pSpCas9(BB)-2A-Puro (PX459) V2.0 Addgene Cat# 62988 (Continued on next page) Cell Reports 43, 114464, July 23, 2024 19
Techniques: Western Blot, Two Tailed Test
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